ÖNSÖZ Testis torsiyonu 25 yaşın altında 1/4000 sıklıkla gözlenen acil bir durumdur. Bu durum ...

17 Pages·2016·0.58 MB·Turkish

ÖNSÖZ

Testis torsiyonu 25 yaşın altında 1/4000 sıklıkla gözlenen acil bir durumdur. Bu
durum infertilite sonuçlanabilen testikuler hasara neden olmasından dolayı acil teşhis
edilip tedavi edilmesi gerekmektedir. Testikuler hasarın patolojik mekanizması
kısmen anlaşılmış olup, reaktif oksijen moneküllerin fazla üretilmesi hücre içi ve doku
hasarında ana etken olduğu gösterilmiştir. Alpha-lipoic acid, quercetineve melatonin
gibi antioksidan moneküller iskemi-reperfüzyon hasarına karşı koruyucu olduğu
gösterilmesine rağmen toksik yan etkileri nedeniyle klinik kullanımda olan monekül
yoktur.Mannitol Üroloji’de(parsiyelnefrektomi) ve Nöroşiruji’de(intracranial
hemorajide) klinik kullanımı olan aynı zaman antioksidan özelliği bilinen moneküldür.
Biz Namık Kemal Üniversite Bilimsel Araştırma Proje birimi tarafından destek alarak,
sıçanlarda deneysel olarak oluşturulan testis torsiyonu modelinde mannitolun
koruyucu etkisini araştıran projeyi sonuçlandırdık.

1
İÇİNDEKİLER

1. Önsöz 1
2. İçindekiler 2
3. Tablolar ve Figürler 3
4. Özet 4
5. Abstract 5
6. Introduction 6
7. Material and Methods 6
8. Experimental Design 6
9. Statistical Analysis 8
10. Results 8
11. Comment 13
12. Conclusions 15
13. References 15

2
Tablolar ve Figürler

Table 1- Experimental groups in study.
Table 2- Histopathologicalevaluation of studygroups
Figure 1.Light microscopy of testicular tissue in different groups.H&E
Figure 2.Light microscopy of testicular tissue in different groups. PCNA
Figure 3-Biochemicalevaluation of anti-oxidantenzymes in studygroups

3
MANNITOLUN TESTİS TORSİYONU ÜZERİNE KORUYUCU
ETKİSİ. BİR DENEYSEL ÇALIŞMA

Giriş: Testikuler torsiyon testis hasarına sebep olan acil bir durumdur. Testis
torsiyonunun zarar verici etkisini azaltan herhangi tedavi seçeneği hastanın geleçek
hayatı için önemlidir. Bu deneysel çalışmada biz sıçan testis torsiyon modelindeki
iskemi-reperfüzyon hasarına mannitolun koruyucu etkisini araştırmayı planladık.
Materyal, Metod:Toplam 32 erkek Spraguee-Dawley sıçan çalışmaya alındı. Her
grupta 8 sıçan olmak üzere 4 grup oluşturuldu.Grup A, sağ testis scrotal insizyonla
dışarı çıkarılıp torsiyon oluşturulmadan tekrar scrotuma yerleştirilen sham grubuydu.
Grup B, sağ testis 720⁰ saat yönünde döndürülerek torsiyon oluşturulan ve tedavi
almayan gruptu. Grup C, benzer şekilde torsiyon oluşturulup torsiyon sonrası saline
tedavisi verilen gruptur. Grup D ise torsiyon sonrası mannitol tedavisi verilen
gruptur.Testisler 3 saat sonar detorsiyone edildikten sonra2 saat sonar orşiektomi
edildi.
Sonuçlar:Testis yapısı mannitol tedavisi almayan torsiyon gruplarında ciddi şekilde
zarar gördü. Öbür yandan, testis dokusunun yapısı koruyucu etki gösteren mannitol
tedavisinde belirgin olarak daha iyiydi. Proliferating cell nuclear antigen(PCNA) index
ve antioxidant aktivite benzer şekilde mannitol grubunda tedavi almayanve saline
grubuna gore yüksekti(p<0.01). Apoptotic index mannitol grubunda tedavi almamış
ve saline grubuna gore belirgin şekilde düşüktü(p<0.01).
Tartışma: Mannitol tedavisi almamış testis torsiyonunda seminifer tubul yapısı ciddi
şekildebozulmasına ragmen mannitol grubunda yapısal bozukluk belirgin olarak azdı.
Mannitol tedavisinin reaktif oksijen seviyesinide belirgin şekilde azalması
apoptozisinde azalmasını sağlayabildi.Bu sonuçlar, diğer organ modellerinde
gösterildiği gibi iskemi-reperfüzyon hasarına mannitol tedavisinin koruyucu etkisini
gösteren çalışmalarla benzerdir.
Sonuç:Mannitol infüzyonunun, sıçanlarda testis torsiyonu sonrası iskemi reperfüzyon
hasarına karşı koruyucu etkisi vardır. Bu deneysel çalışmanın sonuçları insanlarda
testis torsiyonunda mannitolun etkinliğini araştırmayı düşünen klinikçilere yol
gösterebilir.
Keywords:Testiculartorsion, Mannitol, IschemiaReperfusionInjury, Treatment, Rat
Model.
Anaktar Kelimeler: Testis torsiyonu, Mannitol, İskemi-reperfüzyon hasarı, Tedavi,
Sıçan modeli

4
MANNITOL HAS PROTECTIVE EFFECT ON TESTICULAR
TORSION. AN EXPERIMENTAL RAT MODEL

Introduction: Testicular torsion is an emergency condition that causes testicular
injury. Any treatment opportunity reducing the destructive effect of testicular torsion is
important for future life of patients. In this experimental study, we aimed to
investigate the protective effect of mannitol on I/R injury in rat testes torsion model.
Material, Methods: A total of 32, male SpragueeDawley rats were included to
survey. Four experimental groups were formed of each including 8 rats. Group A was
a sham group in which the right testis was brought out through a scrotal incision and
then replaced to the scrotum without torsion. In Group B, right testis was torsioned by
rotating 720⁰ clock-wise and fixed to scrotum without any treatment. In Group C,
same testicular torsion process was performed with saline infusion just after testicular
torsion. In group D, mannitol infusion was used just after testicular torsion. Testicles
were detorsioned after 3 hours and left inside for more 2 hours before orchiectomy.
Histopathological, immunohistochemical and biochemical analysis were performed.
Results: Testicular architecture was significantly disturbed in torsion groups without
mannitol infusion. On the other hand, testicular tissue structure was significantly
better in mannitol treated group, which demonstrated a protective effect. Similar
findings were also shown for proliferating cell nuclear antigen(PCNA) index and
antioxidant activity which were higher in mannitol group compared to no treatment
and saline groups.(p<0.01) Apoptotic index was also significantly decreased in
mannitol treated group compared to no treatment and saline groups.(p<0.01)
Discussion: The seminiferous tubule structure in testicular torsion without mannitol
treatment was significantly disturbed whereas the structural disruption was
considerable less in mannitol group. Mannitol treatment had also significantly
decreased reactive oxygen radical levels and was able to decrease apoptosis. The
results were consistent with other organ model studies that evaluated the protective
effect of mannitol treatment in ischemia-reperfusion injury.
Conclusion: Mannitol infusion had protective effect against ischemia reperfusion
injury in testicular torsion at rats. This experimental study results may guide the
clinicians to evaluate the effectiveness of mannitol in human testicular torsion.
Keywords:Testiculartorsion, Mannitol, IschemiaReperfusionInjury, Treatment, Rat
Model.

5
Introduction
Testicular torsion is an emergency condition with an incidence of 1/4000 in males
under 25 years of age.[1Williamson, 1976] It causes testicular injury, leading to
potential of infertility and subfertility; thus, immediate diagnosis and intervention is
mandatory.[2 Çay, 2006] Although the main pathological mechanisms of testicular
injury in torsion has partially been understood, overproduction of reactive oxygen
species(ROS) was implicated as one of the main factors in cellular and tissue
damage.[3Dokmeci, 2006] Several antioxidant molecules like alpha-lipoic acid,
quercetine and melatonin were found to be effective against ischemia-
reperfusion(I/R) injury, but these molecules were unfavourably used in clinical
practice because of their toxic side effects.[4-6Aktoz,2010; Kanter, 2010; Ozbal S,
2012]
Mannitol has traditionally been administered before partial nephrectomy to reduce
ischemic renal damage as an intravascular volume expander with a function of free
radical scavenging, as well as being an osmotic diuretic.[7Zager, 1985] It reduces
oxidant-derived injury in kidneys, heart and lungs.[8-10England, 1986;Haraldsson,
1995;Weinbroum, 1999] In this experimental study, we hypothesized that mannitol
may have antioxidant protective effect on ischemia and reperfusion injury in testicular
torsion and aimed to investigate this protective effect with histopathological and
biochemical outcomes in rat testes.
MaterialandMethods
With the approval of local animal care and use committee, a total of 32, 6-months
mature male SpragueeDawley rats, weighting between 240 to 280gr. were included
to study. We randomly formed four experimental groups, of each including 8
rats(Table-1). All animals were housed at a temperature and light controlled room,
with ad libitum access to water and rat chow. All animals received human care
according to the criteria outlined in the ‘‘Guide for the Care and Use of Laboratory
Animals’’.
Experimental Design
Surgical procedures were performed under ketamine(50mg/kg,i.p.) and
xylazine(10mg/kg,i.p.) anaesthesia with sterile conditions. A scrotal midline incision
was made and torsion was induced by rotating the right testis 720⁰ clock-wise and
maintained by fixing the testis. Same surgical procedure was performed in sham
group except testicular torsion. Saline(NaCl at 0.09%,10 mL/kg/min) was
administered during the procedure to all groups for hydration. After 3 hours torsion
period, the suture was removed with de-torsion procedure. Testis was replaced into
the scrotum for an extra 2 hours period. At the end of study, the rats were
decapitated and right orchiectomy was performed for biochemical and
histopathological examinations.(Table 1)

6
Sham group (Group A): A sham procedure was performed to determine the
biochemical and histopathologic basal values. The right testis was brought
out through the incision and then replaced to the scrotum without torsion. A
4/0 silk suture was used to fix the testis into the scrotum. After 2 hours
period, right testis was removed for evaluation.
Ischemia-reperfusion (I-R)/untreated group (Group B): After 3 hours of
unilateral testicular torsion, de-torsion was performed and testis was replaced
to scrotum and fixed. The rats in that group only received saline solution
(NaCl at 0.02%, 10 mL/kg/min) during the procedure and did not receive any
treatment after detorsion process. After 2 hours of detorsion period, right
testis was removed for evaluation.
Ischemia-reperfusion (I-R)/saline bolus treated group (Group C): The
same surgical procedure (torsion and detorsion) as in group 2 was
performed. The rats were given a bolus of saline solution (1 mg/kg;i.v.)
immediate after the detorsion. The bolus injection was given intravenously in
2 minutes period and the testis was removed 2 hours after detorsion.
Ischemia-reperfusion (I-R)/mannitol bolus treated group (Group D):The
same surgical procedure (torsion and detorsion) as in other groups was
performed. The rats were treated with a bolus of mannitol (1 mg/kg;i.v.)
immediate after the detorsion. The bolus injection was given intravenously in
2 minutes period that started immediately after reperfusion and the testis was
removed 2 hours after the de-torsion.

Table 1- Experimental groups in study.

Testicular specimens were individually immersed in Bouin’s fixative, dehydrated in
alcohol and embedded in paraffin. Sections of 5µm were obtained, de-paraffinized,
and stained with hematoxylin and eosin(H&E) for evaluation by a histologist in a
random order under blindfold conditions with standard light microscopy by the same
pathologist. Three slides, prepared from the upper, lower and mid-portions of the
testes. Mean seminiferous tubule diameter(MSTD) was measured in micrometers.
Spermatogenesis was assessed histopathologically using Johnsen’s mean testicular
biopsy score(MTBS) criteria.[3,4Dokmeci, 2006; Aktoz,2010] A score of 0–10 was
given to each tubule according to epithelial maturation. Preparations were evaluated
with a bright field microscope(Olympus CX41-Japan) and photographed. We
determined testicular tissue antioxidant enzyme activities including catalase(CAT),
superoxide dismutase(SOD) and glutathione peroxidase(GSH-Px). Testicular tissue
levels of lipid peroxidation products; malondialdehyde(MDA) and xanthine
oxidase(XO) activity were also determined.
7
Immunohistochemical reactions were performed according to the avidin biotin
complex technique described by Hsu et al. [11Hsu, 1981]. The sections were
incubated with specific monoclonal anti PCNA antibody (Cat. #MS-106-B, Thermo
LabVision, USA).To quantify the incidence of PCNA, ten seminiferous tubules were
counted in each slide. Both stained and non-stained germ cells were counted, and
the ratio of stained cells to the total number of germ cells, “PCNA index,” was
calculated for each seminiferous tubule.
Germ cell apoptosis was evaluated by the terminal dUTP nick end-labeling (TUNEL)
assay. The TUNEL method, which detects fragmentation of DNA in the nucleus
during apoptotic cell death in situ, was employed using an apoptosis detection kit
(TdT-FragelTM DNA Fragmentation Detection Kit, Cat. No. QIA33, Calbiochem,
USA). Quantitative analysis of testicular apoptosis was estimated. The incidence of
apoptosis was evaluated in 100 tubules of each testis. These seminiferous tubules
containing three or more apoptotic cells by TUNEL stain were calculated. The
apoptosis percentage was calculated by the ratio of the positive seminiferous tubules
of apoptosis to the total number of seminiferous tubules in a cross-section.
Frozen testis tissue was homogenized. The homogenates were filtered and
centrifuged using a refrigerated centrifuge at 4 ⁰C and supernatant were frozen at -20
⁰C in aliquots until used for biochemical assays. The protein content of the
supernatant was determined using the Lowry method[12]Catalase, SOD, GSH-Px
and MDA level were determined by usingRat ELISA Kit(SRB/Shanghai).
Measurement of serum XO activity was measured spectrophotometrically by the
formation of uric acid from xanthine through an increase in absorbance at 293
nm.[13] The results were expressed in units per liter of plasma(U/L).
Statistical Analysis
All data were analyzed with the Statistical Package for the Social Sciences for
Windows software(Version 17.0 SPSS, Chicago, IL). Data were presented as mean
and standard deviation or percentage. Data in independent groups were analyzed for
normalcy with Kolmogorov–Smirnov test and further evaluated with independent t-
test or Mann–Whitney U test. Data in dependent groups were analyzed with paired t-
test or Wilcoxon signed test after evaluation of normalcy with Kolmogorov–Smirnov
test.
RESULTS
The mean MSTD and Johnsen’s MTBS values in each group are shown in table 2.
Mean MSTD and MTBS of the Group B(I/R) and Group C(I/R+saline) were
significantly lower than Group D(I/R + mannitol) and sham group(p< 0.01).

8
Histopathological Evaluation
Apoptotic Index PCNA Index MTBS MSTD
Group A 3,88±0,83b 36,47±1,36b 9,23±0,17b 273,25±5,14b
Group B 25,88±2,91a,b 22,22±2,34a,b 4,57±0,22a,b 209±6,11a,b
Group C 25,52±2,87a,b 22,51±2,99a,b 4,76±0,14a,b 210,63±5,09a,b
Group D 15,25±1,48a 29,28±1,66a 6,72±0,25a 229,63±6,18a

Table 2 - Histopathological evaluation of study groups
MSTD; Mean seminiferous tubular diameter, MTBS; Mean testicular biopsy
score,
PCNA; Proliferating cell nuclear antigen, I/R; Ischemia- reperfusion,
Group A; Sham operation group, Group B (I/R); Three hours ischemia and two
hours reperfusion, Group C (I/R+Saline); Three hours ischemia and two hours
reperfusion and saline bolus treated group, Group D (I/R+Mannitol); Three hours
ischemia and two hours reperfusion and mannitol bolus treated group.
a P < 0.01 compared to group A
b P < 0.01 compared to group D

Testicular architecture was normal in Sham group. Seminiferous tubular morphology
was regular with normal spermatogenesis(Fig. 1a). Microscopic findings of testicular
tissue were similar in group B(I/R) and group C(I/R+Saline). These findings were a
marked decrease in the seminiferous tubular diameter, with severe distortion,
extensive disorganization, sloughing, and a loss of maturation of germ cells(Fig. 1b).
The tissue appearance was significantly better in Mannitol group with improved
histological findings compared to Group B and Group C(Fig. 1c).

9
Figure 1.Light microscopy of testicular tissue in different groups. H&E: (a)In sham
group, normal testicular architecture was seen; (b)After I/R, severe testicular damage
was noted; (c)Mannitol treatment prevented testicular damage. (H&E, scale bar, 50
µm).

Under light microscopy, I/R and I/R+Saline groups had similar immunohistochemical
findings. There were significant number of PCNA positive cells in sham group(Fig.
2a). The number of PCNA positive germinal cells was lower in the I/R and
I/R+Salinegroups(Fig. 2b). PCNA positive germinal cells were also lower in mannitol
treated group, compared to sham group(Fig. 2c), but the mean PCNA index of this
group was significantly higher than Group B and Group C(p < 0.01)
The amount of germ cell apoptosis was higher in I/R and I/R+Salinegroups(Fig. 2e)
compared to sham group(Fig. 2d). Mannitol infusion markedly reduced the numberof
germ cell apoptosis.(Fig. 2f)The apoptotic index significantly decreased in mannitol
treated I/R group, compared to I/R and I/R+Saline groups.(p < 0.01)
Oxidant(MDA, XO) and antioxidant(GPx, CAT and SOD) levels of the experimental
groups are shown in Fig 3. The SOD, CAT and GPx activities in I/R+Mannitol group
were significantly higher than those in the I/R group(p<0.01). Malondialdehyde levels
in testes were significantly higher in I/R group compared to the sham and the
I/R+Mannitol group.(p < 0.01) Xantine Oxidase activity in I/R+Mannitol group were
significantly lower than those in the I/R group, but was higher than control group.( p <
0.01)

10